RNA interference (RNAi) is a phenomenon wherein double-stranded RNA, when present in a cell, inhibits expression of a gene that has an identical or nearly identical sequence. Inhibition is caused by degradation of the messenger RNA (mRNA) transcribed from the target gene1. The double-stranded RNA responsible for inducing RNAi is termed interfering RNA. The mechanism and cellular machinery through which dsRNA mediates RNAi has been investigated using both genetic and biochemical approaches. Biochemical analyses suggest that dsRNA introduced into the cytoplasm of a cell is first processed into RNA fragments 21–25 nucleotides long2,3,4,5,6. It has been shown in in vitro studies that these dsRNAs, termed small interfering RNAs (siRNA) are generated at least in part by the RNAse III-like enzyme Dicer7. These siRNAs likely act as guides for mRNA cleavage, as the target mRNA is cleaved at a position in the center of the region covered by a particular siRNA8. Biochemical evidence suggests that the siRNA is part of a multicomponent nuclease complex termed the RNA-induced silencing complex (RISC)2. One of the proteins of this complex, Argonaute2, has been identified as a product of the argonaute gene family9. This gene family, which also contains the C. elegans homolog rde-1 and related genes, the N. crassa homolog qde-2, and the Arabidopsis homolog arg-1, has been shown to be required for RNAi through genetic studies10,11,12. Genetic screens in C. elegans have also identified the mut-7 gene as essential for RNAi. This gene bears resemblance to RNAse D, suggesting that its gene product acts in the mRNA degradation step of the reaction13.
Although the use of easily manipulated model systems such as C. elegans and D. melanogaster in gene function studies can yield clues concerning possible new drug targets in mammals, a more direct approach would be to study gene function in mammalian model systems. It has previously been demonstrated that dsRNA can be used to induce RNAi and inhibit target gene expression in mouse oocytes and early embryos14,15. However, data obtained in a number of other studies have indicated that the use of dsRNA to induce RNAi in cultured mammalian cells or post-embryonic tissue may not be effective as a sequence-specific method of gene silencing16,17. This discrepancy may be due in large part to the well-documented dsRNA-mediated induction of interferon synthesis, a response pathway not present in oocytes and early embryos. Activation of dsRNA dependent enzymes leads to non-sequence specific effects on cellular physiology and gene expression18,19,20,21. A major component of the interferon response is the interferon-induced dsRNA-dependent protein kinase, protein kinase R (PKR), which phosphorylates and inactivates the elongation factor eIF2a. In addition, interferons induce the synthesis of dsRNA dependent 2-5(A) synthetases, which synthesize 2′-5′ polyadenylic acid leading to the activation of the non-sequence specific RNAse L22.
The PKR pathway however, is not activated by dsRNA of less than 30 base pairs in length and full activation requires dsRNAs 80 base pairs in length19,20. This fact suggested that if siRNAs are used to initiate RNAi instead of longer dsRNAs, it would be possible to circumvent at least part of the interferon response. Data obtained from studies in which siRNA 21–25 base pairs in length was delivered to mammalian cells in culture indicated that sequence-specific inhibition through RNAi is indeed effective23,24. In these studies, gene-specific inhibition was observed in a variety of both immortalized and primary cell lines. The degree of inhibition varied between 80–96% using siRNA targeted against a reporter gene expressed from transiently transfected plasmids containing strong enhancers. Expression of a control reporter gene of unrelated sequence was unaffected by the siRNA, and no inhibition was observed using siRNAs against unrelated sequences. Expression of endogenous genes could also be inhibited to levels below detection by siRNA. These data demonstrate the specificity and effectiveness of siRNA-mediated RNAi in cultured mammalian cell lines and suggest that the interferon response is not activated by siRNAs of this length. These results suggest that RNAi can indeed be used to effectively inhibit gene expression in mammalian cells.
The ability to specifically inhibit expression of a target gene by RNAi has obvious benefits. For example, many diseases arise from the abnormal expression of a particular gene or group of genes. RNAi could be used to inhibit the expression of the deleterious gene and therefore alleviate symptoms of a disease or even provide a cure. For example, genes contributing to a cancerous state or to viral replication could be inhibited. In addition, mutant genes causing dominant genetic diseases such as myotonic dystrophy could be inhibited. Inflammatory diseases such as arthritis could also be treated by inhibiting such genes as cyclooxygenase or cytokines. Examples of targeted organs would include the liver, pancreas, spleen, skin, brain, prostrate, heart etc. In addition, RNAi could be used to generate animals that mimic true genetic “knockout” animals to study gene function.
Drug discovery could also be facilitated by siRNA technology. The siRNA approach for target validation will provide a quicker and less expensive approach to screen potential drug targets. Information for drug targeting will be gained not only by inhibiting a potential drug target but also by determining whether an inhibited protein, and therefore the pathway, has significant phenotypic effects. For example, inhibition of LDL receptor expression should raise plasma LDL levels and, therefore, suggest that up-regulation of the receptor would be of therapeutic benefit. Expression arrays can be used to determine the responsive effect of inhibition on the expression of genes other than the targeted gene or pathway25. It will place the gene product within functional pathways and networks (interacting pathways).
The efficient delivery of biologically active compounds to the intracellular space of cells has been accomplished by the use of a wide variety of vesicles. One particular type of vesicle, liposomes, is one of the most developed types of vesicles for drug delivery. Liposomes, which have been under development since the 1970's, are microscopic vesicles that comprise amphipathic molecules which contain both hydrophobic and hydrophilic regions. Liposomes can be formed from one type of amphipathic molecule or several different amphipathic molecules. Several methods have been developed to complex biologically active compounds with liposomes. In particular, polynucleotides complexed with liposomes have been delivered to mammalian cells. After the description of DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride)26, a number of cationic lipids have been synthesized for this purpose. Essentially all the cationic lipids are amphipathic compounds that contain a hydrophobic domain, a spacer, and positively-charged amine(s). The cationic lipids are sometimes mixed with a fusogenic lipid such as DOPE (dioleoyl phosphatidyl ethanolamine) to form liposomes. The cationic liposomes are then mixed with plasmid DNA and the binary complex of the DNA and liposomes are applied to cells in a tissue culture dish or injected in vivo. The ease of mixing the plasmid DNA with the cationic liposome formulation, the ability of the cationic lipids to complex with DNA and the relative high levels of transfection efficiency has led to increasing use of these formulations. However, these cationic lipid formulations have a common deficiency in that they are typically toxic to the cells in culture and in vivo.2728. More recently lipids have been used in association with other DNA-binding compounds to facilitate transfection of cells. The present invention provides new amphipathic compounds, and methods of preparation thereof, to be used to prepare novel complexes of biologically active polyions for delivery to animal cells in vitro and in vivo. The complexes facilitate high efficiency transfer of the polyion from outside the cell to the inside a cell with low toxicity.
The present invention describes transfection reagents and methods to deliver siRNA to animal cells in vitro and in vivo with high efficiency and low toxicity. We demonstrate that our method effectively delivers siRNA to animal cells for the purpose of RNA interference.